The present invention relates to novel tumor antigen proteins and genes encoding the same, and antibodies against said tumor antigen proteins, as well as methods of treating, preventing, or diagnosing tumors using such materials.
It is known that the immune system, particularly T cells, plays an important role in vivo in tumor rejection. Indeed, infiltration of lymphocytes having cytotoxic effects on tumor cells has been observed in human tumor foci (Arch. Surg., 126:200-205, 1990), and cytotoxic T lymphocytes (CTLs) recognizing autologous tumor cells have been isolated from melanomas without great difficulties (e.g., Immunol. Today, 8:385, 1987; J. Immunol., 138:989, 1987; and Int. J. Cancer, 52:52-59, 1992). In addition, the results of clinical treatment of melanomas by T cell introduction also suggest the importance of T cells in tumor rejection (J. Natl. Cancer. Inst., 86:1159, 1994).
Although it has long been unknown about target molecules for CTLs attacking autologous tumor cells, the recent advance in immunology and molecular biology has gradually revealed such target molecules. Specifically, it has been found that using T cell receptors (TCRs), CTL recognizes a complex between a peptide, called tumor antigen peptide, and a major histocompatibility complex class I antigen (MHC class I antigen, and in the case of human, referred to as HLA antigen), and thereby attacks autologous tumor cells.
Tumor antigen peptides are generated from proteins specific for tumors, that is, tumor antigen proteins. Thus, the proteins are intracellularly synthesized and then degraded in cytoplasm into the peptides by proteasome. On the other hand, MHC class I antigens (HLA antigens) formed at endoplasmic reticulum, when bind to the above tumor antigen peptides, are transported via cis Golgi to trans Golgi, i.e., the mature side and carried to the cell surface where they are presented as an antigen. A tumor-specific CTL recognizes this complex presented as an antigen, and exhibits its anti-tumor effects through the cytotoxic effect or production of lymphokines (Rinsho-Menneki, 27(9):1034-1042, 1995). As a consequence of such elucidation of a series of actions, it has become possible to treat tumors by using tumor antigen proteins or tumor antigen peptides as so-called cancer vaccines which enhance tumor-specific CTLs in a patient.
As such tumor antigen proteins, T. Boon et al. identified a protein named MAGE from human melanoma cells for the first time in 1991 (Science, 254:1643-1647, 1991), and thereafter several additional tumor antigen proteins have been identified from melanoma cells.
As reviewed by T. Boon et al. (J. Exp. Med., 183, 725-729, 1996), tumor antigen proteins hitherto identified can be divided into the following four categories.
Tumor antigen proteins belonging to the first category are those which are expressed in testis only as normal tissues, while they are expressed in melanoma, head and neck cancer, non-small cell lung cancer, bladder cancer and others, as tumor tissues. Among tumor antigen proteins in this category are the above-described MAGE and analogous proteins constituting a family of more than 12 members (J. Exp. Med., 178:489-495, 1993), as well as BAGE (Immunity, 2:167-175, 1995) and GAGE (J. Exp. Med., 182:689-698, 1995), all of which have been identified in melanoma cells.
Although some of such tumor antigen proteins in this category are highly expressed in melanoma, the expression thereof is observed in only 10 to 30% of patients having a particular tumor other than melanoma, and therefore, they cannot be applied widely to treatments or diagnoses of various tumors.
Tumor antigen proteins belonging to the second category are those which are expressed only in melanocytes and retina among normal tissues, and in melanomas among tumor tissues. Since these tissue-specific proteins are highly expressed in melanomas, they would function as tumor antigen proteins specific for melanomas, Among tumor antigen proteins in this category are tyrosinase (J. Exp. Med., 178:489-495, 1993), MART-1 (Proc. Natl. Acad. Sci. USA, 91:3515, 1994), gp100 (J. Exp. Med., 179:1005-1009, 1994), and gp75 (J. Exp. Med., 181:799-804, 1995). Genes encoding these proteins have all been cloned from melanoma cells. Melan-A (J. Exp. Med., 180:35, 1994), which has been separately isolated, has proved to be identical with MART-1.
However, the tumor antigen proteins of this category cannot be used widely in the treatments or diagnoses of various tumors, since they are not expressed in tumors other than melanoma.
Tumor antigen proteins belonging to the third category are those which are expressed as tumor antigen peptides recognized by CTL as a result of tumor-specific mutations. Among tumor antigen proteins in this category are mutated CDK4 (Science, 269:1281-1284, 1995), xcex2-catenin (J. Exp. Med., 183:1185-1192, 1996), and MUM-1 (Proc. Natl. Acad. Sci. USA, 92:7976-7980, 1995). In CDK4 and xcex2-catenin, a single amino acid mutation increases the binding affinity of the peptides to MHC class I antigen, which allows them to be recognized by T cells. In MUM-1, an intron, which normally is not translated, is translated due to mutation, and the resultant peptide is recognized by T cells. However, since such mutations occur at low frequency, they cannot be applied widely to treatments or diagnoses of various tumors.
Tumor antigen proteins belonging to the fourth category are those widely expressed in normal tissues and also recognized by CTL, example of which includes P15 identified from melanoma cells (J. Immunol. 154:5944-5955, 1995).
Some of known tumor antigen proteins as described above are expressed only in limited tumors such as melanoma, and the others are expressed only in a small number of patients having a particular tumor even if they are expressed in various kinds of tumor, and therefore, they cannot be used widely in the treatments or diagnoses of various tumors. Accordingly, it is necessary to identify a tumor antigen that is widely applicable to various tumors, for instance, squamous cell carcinomas (e.g., esophageal cancer, lung cancer), which show much higher incidence than melanomas, before the application of tumor antigen proteins or tumor antigen peptides generated therefrom by intracellular degradation to the treatments or the diagnoses of various tumors. In this connection, the present inventors have conducted cloning of a gene encoding a tumor antigen protein from squamous cell carcinoma cells derived from esophageal cancer, and, for the first time, succeeded in cloning a gene encoding a tumor antigen protein (SART-1) from tumor cells other than melanomas (International Publication WO 97/46676).
Regarding diagnosis of tumors, various approaches are now being made by means of antibodies against tumor associated antigens, which, for example, include detection of antigenic substances in blood by radioimmunoassay, ELISA, or the like; histological cytodiagnosis by immunohistochemical methods such as enzyme-linked immunosorbent assay or fluorescent antibody technique; or imaging of tumors (Fishman, W. H. et al., xe2x80x9cOncodevelopmental Markersxe2x80x9d, Academic Press, 1983). However, leading tumor markers currently used give a false positive in benign diseases with relatively high frequency. Therefore, there has been a great demand for the identification of a tumor antigen protein highly specific for tumors and the isolation of antibodies against the same to be used in diagnosis.
Furthermore, when using a particular tumor antigen protein or tumor antigen peptide as, for example, a vaccine, it is desired that a patient who expresses the tumor antigen protein and is possibly reactive to treatment with the tumor antigen protein/peptide is diagnosed and selected before applying the protein/peptide to the patient. Tumor antigen proteins with high tumor specificity or antibodies directed thereto are believed to be extremely useful diagnostic agents in the selection of such subject patients. From this point of view, it is desired to identify a tumor antigen protein having higher tumor specificity and is applicable to a wide range of tumor patients, and also an antibody raised against the same.
One of purposes of the present invention is to provide a novel tumor antigen protein or gene, or antibodies against the tumor antigen protein. It is also a purpose of the invention to provide a method of treating, preventing, or diagnosing tumors using such substances. More particularly, the present invention aims to provide a tumor antigen protein highly specific for tumors or corresponding tumor antigen peptides, DNAs encoding them, and antibodies recognizing and binding them, all of which are widely applicable to treatment or diagnosis of various tumors, especially squamous cell carcinomas.
To this end, the present inventors established a squamous cell carcinoma cell line KE-4 derived from esophageal cancer (hereinafter referred to as an esophageal cancer cell line KE-4 or simply as KE-4), and also established CTL (hereinafter referred to as KE-4CTL) which recognizes tumor antigen peptides restricted to HLA-A2601 and HLA-A2402 which are MHC class I antigens expressed in said KE-4 (Cancer Res., 55:4248-4253, 1995).
Fibroblast cell line VA-13 cells were then co-transfected with a recombinant plasmid of cDNA library prepared from KE-4 and a recombinant plasmid containing HLA-A2601 cDNA. The transfectants were then treated with KE-4CTL and screened for the activation of KE-4CTL by measuring the amount of IFN-xcex3 produced. After carrying out the screening repeatedly, the inventors have succeeded in cloning a gene encoding a novel tumor antigen protein. The nucleotide sequence of the cloned gene is shown in SEQ ID NO: 1.
The present inventors then inserted the gene encoding a novel tumor antigen protein into a plasmid vector for expressing a fusion protein with GST, transformed E. coli cells with the resultant vector, and prepared a fusion protein between GST and the novel tumor antigen protein of the present invention. Various cell lines and tissues were analyzed by Western blot analysis using antibodies obtained by immunizing a rabbit with the above fusion protein. As a result, expression of the novel tumor antigen protein of the present invention having a molecular weight of about 43 kilo Dalton (kD) was observed in 100% of head and neck squamous cell cancers, 60% of esophageal squamous cell cancers, 50% of lung squamous cell cancers, and 50% of lung adenocarcinomas examined, although no expression was observed in any of all normal tissues except for testis and fetal liver, melanomas, and leukemia. It was also demonstrated that cancer cells which were positive in the Western blot analysis (cancer cells expressing the tumor antigen protein of the present invention) are indeed recognized and damaged by tumor specific CTLs.
As described above, since the tumor antigen protein of the present invention is expressed specifically and with high frequency in various squamous cell cancers and adenocarcinomas, it should be useful as a pharmaceutical for activating the antitumor immunity of patients suffering from such cancers. Further, antibodies against the tumor antigen protein of the present invention should be effectively used in diagnosis of cancer patients and selection of subject patients.
The nucleotide sequence of DNA encoding about 43 kDa tumor antigen protein of the present invention corresponds to the nucleotide sequence beginning at position 1517 of DNA encoding the tumor antigen protein, SART-1, which is described in the International Publication WO 97/46676 (shown as SEQ ID NO: 2 in WO 97/46676). However, the tumor antigen protein of the present invention is believed to be expressed in vivo (in tumor tissues or tumor cells) independently of SART-1, because it was detected in various tumor tissues and tumor cells as a protein having a molecular weight of about 43 kDa in the above-mentioned Western blot analysis and, further, it shows an expression pattern inconsistent with that of SART-1 presumably having a molecular weight of about 125 kD.
The present invention was established on the basis of these findings.
Thus, the present invention relates to a DNA (a) or (b) below:
(a) a DNA consisting of the base sequence shown in SEQ ID NO: 1; or
(b) a DNA that hybridizes under stringent conditions to a DNA consisting of the base sequence shown in SEQ ID NO: 1 and that encodes a protein having activity as a tumor antigen.
The present invention also relates to expression plasmids containing said DNA, tumor antigen proteins obtainable through expression of said DNA, antibodies that recognize said tumor antigen proteins, and use thereof.